凝胶电泳微流控芯片提取外泌体及对人血浆外泌体中miRNA-21的测定

Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma

摘要:

为了开发一种用于人体血浆中外泌体的高效快速提取和分离的新型微流控芯片，文中收集健康人体外周血液样本，自主设计并制备基于纳米多孔薄膜和琼脂糖凝胶电泳的微流芯片。提取的外泌体使用透射电镜、Nanosight和Western blotting等技术进行表征，鉴定并分析其形态、浓度和粒径分布。同时将超速离心法和微流芯片所提取的外泌体进行粒径和浓度的分析比较，探讨两种方法各自的提取效率。最后，利用RT-PCR技术分析外泌体中miRNA-21的相对表达量。 凝胶电泳微流芯片可在1 h内快速的从血浆中高效率地提取出纯度高、大小完整、尺寸分布在30–200 nm之间的外泌体，满足后续下游分析的要求。通过与现有最普遍的超速离心法进行对比分析，当血浆样本量小于100 μL时，凝胶电泳微流芯片提取外泌体的效率为超速离心法的3.80倍。凝胶电泳微流芯片提取外泌体的优化参数是：电场电压：100 V；琼脂糖凝胶浓度：1.0%；注射泵流速：0.1 mL/h。凝胶电泳微流芯片可快速高效地提取出外泌体，对外泌体与癌症生物标记物的相关研究具有潜在的巨大优势，也为基于外泌体的即时诊断技术提供了可能。

Abstract:

We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30–200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.